Over the last decade, considerable effort has focused on the prevention and treatment of aberrant blood clot (thrombus) formation which can result in ischemic events and other complications. Prevention of ischemic events could significantly reduce the prevalence diseases such as of acute myocardial infarction, angina, complications following angioplasty, restenosis, thrombotic stroke, ischemic neurological deficits and other ischemic disorders.
Histological studies of vascular injuries have demonstrated the importance of platelets, adenosine diphosphate (ADP), smooth muscle cells and thrombosis in atherosclerotic cardiovascular, cerebrovascular and peripheral arterial diseases. However, while a variety of anti-thrombotic drugs have been available, the primary mechanisms of platelet aggregation were not well understood and this lack of understanding has prevented the development of additional effective therapeutics for ischemic disease conditions.
Integrins mediate cell adhesion and transduce signals that are critical in the dynamic regulation of cell adhesion, spreading, migration, and proliferation. Thus, integrin activity is central to regulation of platelet adhesion and clot formation (Hynes, 2002 and Ginsberg et al., 2005). Bidirectional signaling of integrins is exemplified in the prototype platelet integrin, αIIbβ3. Inside-out signaling of integrin αIIbβ3 requires talin binding to the cytoplasmic domain of αIIbβ3, which leads to conformational changes, which, in turn, propagate to the extracellular ligand binding domain, activate the ligand binding function (Tadokoro et al., 2003 and Vinogradova et al., 2002). Ligand binding to αIIbβ3 not only forms adhesive bonds, but also induces outside-in signaling leading to platelet spreading, secretion, amplification of platelet aggregation and subsequent clot retraction (Shattil et al., 1998). It is known that outside-in signaling of integrin αIIbβ3 requires phosphorylation of the β3 subunit cytoplasmic domain at Y747 and Y759 (Law et al., 1999). It has also been reported using multiple Src family protein tyrosine kinases-deficient mouse models that Src plays an important role in the integrin outside-in signaling (Obergfell et al., 2002). However, the exact molecular events and the direct requirement for this kinase in β3 tyrosine phosphorylation in human platelets remain to be established.
The cytoplasmic domain of the β3 subunit is critical in integrin bidirectional signaling. Inside-out signaling requires the membrane proximal region of β3, including the highly conserved N744PXY747 motif which directly interacts with the talin head domain allowing receptor activation. It has been shown that the C-terminus of β3 interacts with Src, an interaction that is important in integrin outside-in signaling leading to cell spreading, and that two residues in the β3 cytoplasmic tail, R760 and T762, are necessary for Src binding (Arias-Salgado et al., 2005). Furthermore, deletion of the C-terminal RGT amino acidsequence of β3 abolishes Src binding (Flevaris et al., 2007). However, it was not known which amino acid sequences are sufficient for Src binding. Moreover, in human platelets, the precise functional role of the R760GT762 residues in outside-in signaling was not understood.
Thus, there exists a need in the art to develop materials and methods which can be used to modulate platelet aggregation. The instant disclosure elucidates aspects of integrin β3-Src signaling in platelet cells and discloses compositions to disrupt platelet cell signaling. Thus, the instant disclosure addresses a deficit in the art by providing methods to reduce platelet aggregation by disrupting intracellular signaling in platelets.